Homobifunctional imidoester-modified zinc nano-spindle attenuated hyphae growth of Aspergillus against hypersensitivity responses

Summary Fungi cause various forms of invasive fungal disease (IFD), and fungal sensitization can contribute to the development of asthma, asthma severity, and other hypersensitivity diseases, such as atopic dermatitis (AD). In this study, we introduce a facile and controllable approach, using homobifunctional imidoester-modified zinc nano-spindle (HINS), for attenuating hyphae growth of fungi and reducing the hypersensitivity response complications in fungi-infected mice. To extend the study of the specificity and immune mechanisms, we used HINS-cultured Aspergillus extract (HI-AsE) and common agar-cultured Aspergillus extract (Con-AsE) as the refined mouse models. HINS composites within the safe concentration range inhibited the hyphae growth of fungi but also reduce the number of fungal pathogens. Through the evaluation of lung and skin tissues from the mice, asthma pathogenesis (lung) and the hypersensitivity response (skin) to invasive aspergillosis were least severe in HI-AsE-infected mice. Therefore, HINS composites attenuate asthma and the hypersensitivity response to invasive aspergillosis.


INTRODUCTION
Fungi are widely distributed in the environment, and although the majority of fungi are generally non-pathogenic to humans, 1-3 some cause various forms of the disease. In particular, superficial infestations from fungi have been diagnosed more frequently, with an increasing number of patients reported. [4][5][6] Fungal allergy can contribute to asthma severity and other hypersensitivity diseases, such as atopic dermatitis (AD), which is closely correlated with allergic rhinitis and allergic asthma, which normally occur in children and young adults. 7,8 Aspergillus fumigatus is the most common cause of invasive fungal diseases (IFDs), which constitute a leading cause of morbidity and mortality in recipients with malignant disease undergoing blood, bone marrow, stem cell transplantation, or solid organ transplantation. 9-11 Poorly maintained ventilation and water systems in medical facilities and contamination of instruments are also reported as potential causes of infection. 10 A plethora of fungal co-infections in patients affected and recovering from coronavirus disease 2019 (COVID-19) have been documented worldwide, raising concern for an outbreak of life-threatening fungal complications in patients with COVID-19. 12,13 Oxygen delivery is crucial for intensive care unit patients infected with COVID-19. The oxygen must be both highly sterilized and purified and requires sterile water for humidification. If oxygen is administered without being humidified, it dries out the mucous membrane and damages the lungs' inner lining. 14,15 The phlegm becomes viscous and is difficult to expel. Inhalation of fungi spores by patients weakens the immune system and can lead to infection of the sinuses, lungs, skin, and soft tissue, thrombosis, necrosis, inflammation, and even death, highlighting the need to optimize prophylaxis against invasive fungal infections and increase treatment choices. 16,17 Numerous new prophylaxis and therapeutic approaches have been explored to resist fungi infection. Many bionanomaterial research teams are committed to studying advanced biomedical candidates based on traditional antibiotics, and safety equipment. [18][19][20] Cumulating in vitro evidence suggests that many of these biomaterial interactions result in synergistic antimicrobial effects with antibiotics (a positive side effect). [21][22][23] The proposed general mechanisms for the ability of nanomaterials to overcome antimicrobial resistance include the following: (1) Membrane damage: nanomaterials tend to rupture the cell membrane In this study, we report an approach for attenuating human responses to invasive aspergillosis in a mouse model through building functional nanomaterials for prophylaxis. In vitro study of homobifunctional imidoester-modified zinc nano-spindle (HINS) material has just proved enhanced biocompatibility, antibiotic efficacy, and blood coagulation reduction in the field of antibiotics. Drug resistance development and pathogenicity are associated with biofilm formation by fungal pathogens, 11,29,30 so we wondered whether we could develop a prophylactic measure against pathogenic fungi by promoting nanomaterial composite substances for packaging of daily stuff, construction materials, and medical instruments to attenuate hypersensitivity responses in lung and skin. We found that the 2.5% mixture ratio of HINS and culture medium would decrease the growth of spores, and the 5.0% mixture ratio of HINS effectively inhibited the hyphae growth of fungi for 90 h and even longer under subtropical conditions suitable for fungal growth. Next, to evaluate the histomorphological effects of HINS on lung and skin hypersensitivity mouse models were established by intranasal (i.n.) and topical exposure to A. fumigatus, called homobifunctional imidoester-modified zinc nano-spindle agar-cultured Aspergillus extract (HI-AsE). Through the evaluation of sensitization, dermatitis severity, and histological analysis of lung and skin tissue, we found that asthma pathogenesis (lung) and the hypersensitivity response (skin) to invasive aspergillosis were less severe in HI-AsE-infected mice compared with common agar-cultured Aspergillus extract (Con-AsE)-infected mice. The Con-AsE group showed significantly greater epidermal and dermal thickness than the HI-AsE group. Meanwhile, the HI-AsE group showed less inflammation compared to the Con-AsE group. In this light, we suggest that HINS composites for daily packaging stuff, construction materials, and medical instruments would help decrease pathogenic fungal infection in the lung and skin inflammation induced by i.n. and topical exposure to mold.

Principle of HINS composite against fungal infection
To examine whether HINS composite administered i.n. and i.p. attenuates fungal growth and activity (Scheme 1), we established the culture medium composition for HINS and collected the cultured Aspergillus, Con-AsE, and HI-AsE, respectively ( Figure 1A and Table 1). HINS was prepared through the facile modified hydrothermal method (Table 1). Owing to the stability and biocompatibility of HINS being the main factors of the enhanced efficiency, we used different concentrations (1-10 mg/mL) of HI (DMS) to modify the ZnO NSs for 20 min. To substantiate the analysis of the concentration-effect reaction, the zeta potentials of the HINS were measured ( Figure 1B). The apparent zeta potential of ZnO NSs reached +21 mV, which revealed the incipient instability of our fundamental materials. Moreover, the apparent zeta potential of 1 mg/mL of the modified HINS composite changed to +44 mV, which indicated that HINS-1 gained electrokinetic potential and had good stability. However, as the concentration of HI increased, the solution became sticky, and the apparent zeta potential of HINS-2 was lower than that of ZnO NSs. Moreover, HINS-3 exhibited no significant zeta potential. These changes demonstrated that the energy shift after proper HI modification would provide the composite with more energy and activity ( Figure 1B). Meanwhile, we observed the morphology of the materials using an SEM instrument: The uniform particles of ZnO NSs and the different concentrations of modified HINS could be shown clearly in the SEM images. Compared with the smooth and clean shape of ZnO NSs ( Figure 1C), the HINS seems to be wearing a bridal gown and has a soft surface ( Figure 1D). The susceptibility of HI to hydrolysis indicated that the HI modification could help to protect the modified materials in an independent stable condition. However, when the concentration of HI was too high, the ZnO NSs mingled and aggregated, and their morphology was obscured ( Figure 1E). Thereby, the optimal concentration of modified HI on ZnO NSs is quite important for preparing its conjugation. Meanwhile, the photos ( Figure 1F) recorded the reaction, showing that a high concentration of HI would overreact with the ZnO NSs; 10 mg/mL of HI would dissolve/ release materials out of the solid phase. All these indicated that the relative electron beam density caused the surface modification of HI of ZnO NSs, and the proper surface energy could lead to enhanced applications of materials. Next, the fungi growing in the HINS-treated agar showed a different morphological tendency ( Figure 2). We noticed that Aspergillus grown on the common agar exhibited strong hyphae (Figure 2A) compared with its fragile hyphae on HINS agar ( Figure 2B). Based on the strong hyphae growth and plump spores of Aspergillus, the HINS could attenuate the hyphae growth and lyse the spores of Aspergillus.

HINS attenuates the hyphae growth of fungi
We designed an ingenious in vitro method to study the effect of the HINS-composed substrate. Here, we updated the reported homogeneous spread-plate culture method and the center culture method with a combination: the sterile agar was mixed with the amounts of HINS. The aspergillosis spores were planted in the center of the composite agar; and the growth condition serves as a contrast to natural Aspergillus, and the growth rate of the fungal spores could be calculated by measuring the surface area of the colony.
Here, we traced the growth of fungal colonization (6,000 Aspergillus spores/dish cultured at 35 C for 3 days). We observed the proportion of the fungal colony and found that the growing tendency of the Aspergillus colony was almost entirely inhibited in the HINS-treated agar plate ( Figure 3A). The daily collected photos recorded the hyphae growth of the fungi sensitively and clearly by the Bio-Rad ChemiDoc XPS+, as the tubular cell wall of hyphae was observed as black filiform. Owing to the principle of this image reader model is capturing the filtering light across the object; almost all the wavelengths of light could pass through the hyphae, appearing black in the image. Inversely, the saturated spores would absorb the light and appear white in the image. Furthermore, the gradient color displayed the density of spore and hyphae. By comparing the common nutrition agar (Negative) and the HINS-treated agar (Treated) in Figure 4A, we found that the hyphae of fungi could not grow on the HINS-treated agar. The colonies of the culture are summarized in Figure 3B, and the results indicated the following: (1) The hyphae were growing from the spore and then growing ahead of the spores in the common nutrition agar, which obeys the natural growth; (2) the activity of hyphae would guide the growth of Aspergillus; (3) in the HINS-treated agar, there was barely hyphae growth of fungi. However, the spores would survive by fission for 3 days, and the hyphae would occur as an extension at a slow speed on the surface of gathering spores. In addition, we collected the grown Aspergillus from those two agar media after 96 h of culture and checked their morphology by iScience Article SEM. The results showed that Aspergillus grew strongly in the common agar plate, and numerous hyphae ( Figure 1A) extended over the surface trending growth. However, in comparison, the hyphae growth of Aspergillus was fragile and fewer hyphae ( Figure 1B) existed in the HINS-treated agar plate.

Optimized HINS composite for the antifungal activity
Optimizing the nanomaterials is an essential property for their medical industry applications, especially the inherent toxicological issues and efficiency. To this end, we studied the three different concentrations (mixture rates: 1.0, 2.5, and 5.0%) of HINS in the composed agar (HI-AsE), and photographs recorded the growth under each condition. We monitored the fungal growth condition for 96 h. The diameter of the grown microbiota at 66 h was observed ( Figure 4A). Notice that in all the culture conditions in this study, we increased the seed density to 6,000 Aspergillus spores/dish and set the culture room at 35 C, which would more approximate the weather condition of a tropical/subtropic rainy climate where the fungal would grow much faster. We found that the 2.5% mixture ratio decreased the growth of spores, and the 5.0% mixture ratio was sufficiently effective to inhibit the hyphae growth of fungi for 90 h and even longer, as shown by the growth trend chart ( Figure 4B). The results showed that the higher the mixture ratio of HINS the greater the antifungal efficiency, which has shown persistent inhibition. Based on several studies of the mycotoxin secreted by Aspergillus, the hyphae growth of the fungi is caused by the apical extension tube of mycelium cells, with the release of mycotoxin. 31,32 Therefore, blocking the hyphae growth of fungi would be desired for antifungal activity.

Gene expression pattern for the hypersensitivity responses in HI-AsE
To explore the mechanism by which HINS controls the hyphae growth, development, and viability of A. fumigatus spores, we extracted RNA from Con-AsE and HI-AsE to compare the mRNA expression levels of seven associated genes (tpsA, tpsB, tpsD, gel4, aspA, hsp90, and dprA). To protect itself from external stressors, such as temperature and humidity, A. fumigatus accumulates compatible solutes, such as trehalose. 33 Trehalose is required for stress resistance and long-term viability. TpsA, tpsB, and tpsD encode enzymes involved in trehalose biosynthesis. 34 Their expression is important for the survival iScience Article of Aspergillus conidia. 31,34 In the HI-AsE group, the expression levels of these genes immediately reduced compared with the Con-AsE group, indicating that HINS had a significant inhibitory effect on trehalose synthesis and impacted conidial survival ( Figure 5A). Two distinct morphological changes characterize the germination of conidia: isotropic growth and polarized growth. 35,36 Therefore, we selected gel4 and aspA for isotropic growth and polarized growth of A. fumigatus spores, respectively. 36, 37 We found a decreasing trend of mRNA expression of gel4 and aspA with the increase in HINS concentration, indicating that HINS inhibited the hyphae growth and spore development of Aspergillus ( Figure 5B). We also tested the heat shock-related gene (hsp90) 38 and oxidative stress-related gene (dprA) 39 as control genes, but there was no significant difference across the groups, revealing that HINS did not alter the fungal responses to temperature changes and oxidative stress ( Figures 5C and 5D). These results provide evidence that HINS inhibited the fungal hyphae growth and spore development and reduced its potential antifungal activity.  We wondered whether the theory of the HINS-treated medium preparation would have applications in medical industries. To gain insight into the attenuated pathogenesis of those fungi spores that hyphae growth from the HINS-treated agar, we evaluated the in vivo pathogenicity of Aspergillus (Con-AsE and HI-AsE) in three different mouse models. To evaluate whether HI-AsE still triggered a pathogenic response in the lung tissues of mice, we administered 40 mg of the extract products from Con-AsE or HI-AsE or vehicle to mice by i.p. 3 times for 2 weeks (Day 1, 7, and 14), followed by an i.n. challenge for three consecutive days (Day 14-16). On Day 20, mice were sacrificed for the collection of lung tissue and blood ( Figure 6A). To analyze whether this induction strategy produces hypersensitivity responses in mice, the total IgE levels from mouse serum were measured. HI-AsE-treated mice had significantly lower IgE levels than those in Con-AsE mice ( Figure 6B). As shown in Figure 6B, the mean total IgE level was 25.7 ng/mL in the vehicle group (PBS), 303.0 ng/mL in the Con-AsE group, and 88.5 ng/mL in  iScience Article the HI-AsE group, indicating that the Con-AsE treated group developed a hypersensitivity response in mice. Moreover, H&E staining for analysis of lung histopathology revealed that intratracheal administration of Con-AsE increased peribronchial and parenchymal infiltration of eosinophils in and around the airways, including significant interstitial infiltration of inflammatory cells, alveolar septal thickening, and collapsed alveolar spaces, compared with those of the vehicle and HI-AsE groups ( Figure 6C). PAS staining of the lung is performed to identify goblet cell hyperplasia in the epithelium and submucosal gland hypertrophy. The Con-AsE group had significantly higher levels of airway mucus (mucin staining) compared with the vehicle and HI-AsE-treated groups ( Figure 6D). The inflammatory cell (eosinophil) infiltration and goblet cell production scores were calculated ( Figures 6C and 6D). Con-AsE (a ring of inflammatory cells of more than 4 cells deep) has the most inflammatory cell infiltration compared with the vehicle and HI-AsE groups ( Figure 6E). The peribronchial wall inflammation was evaluated, and no score above 1 point could be noted, suggesting that no tested component in the HI-AsE group induced an inflammatory reaction in the pulmonary tissue. Goblet cell hyperplasia and airway mucus were improved in the Con-AsE group compared with the vehicle and HI-AsE groups (goblet cells were <25% in both), which reflects inflamed epithelium from Con-AsE irritation ( Figure 6F). These findings indicate that we were successful in designing an AsE-induced hypersensitivity mouse model, which is consistent with other studies. [40][41][42] Furthermore, the IgE level from mouse serum in the HI-AsE group indicated no noticeable allergic reactions, and the lung histological analysis was similar to that seen in the vehicle group. In addition, the blood biochemistry analysis (alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine) of mice conducted 14 days after intravenous administration of HINS showed good biocompatibility. These results suggested that HINS-cultured Aspergillus do not have potent pathogenic functions in vivo, which may be due to disrupting the development of its pathogenic toxins. Therefore, HINS could be used effectively and safely in medical devices, particularly those associated with ventilators. The term ''atopic march'' was created to describe the progression of atopic disorders from AD in infants to allergic rhinitis and asthma in children. 7, 43,44 To simulate this complication, the mouse epidermis was exposed to either Con-AsE or HI-AsE 3 times per week while allergic airway inflammation was induced ( Figure 7A). As shown in Figure 7B, the mean total IgE level was significantly lower in the vehicle (19.1 ng/ mL) and HI-AsE groups (50.1 ng/mL) than in the Con-AsE group (322.0 ng/mL). According to H&E staining, eosinophil infiltration was significantly lower in the HI-AsE group than in the Con-AsE group (Figure 7C), and lung PAS staining confirmed that the HI-AsE and control groups had similar levels of airway mucus ( Figure 7D). Based on the quantification of histological staining and scoring criteria, inflammatory cell infiltration in the Con-AsE group (nearly 4 points) was more than that in the vehicle (1 point) and HI-AsE groups (<2 points). Goblet cells produced by the HI-AsE group (<1 point) were almost comparable to the vehicle group ( Figures 7E and 7F). The results are consistent with the theoretical report of the pathogenesis-related protein located on hyphae of fungi. 7, 29 In addition, the Con-AsE group developed significant epidermal dryness and AD symptoms after 20 days, whereas this inflammatory symptom was not observed in the skin of the HI-AsE-or vehicle-treated groups ( Figure 8A). Based on the SCORAD index, the skin of the Con-AsE group had the most noticeable inflammatory score on Day 10, whereas the HI-AsE group had no significant changes in the inflammatory signature over the 3-week treatment period ( Figure 8B). On Day 21, histological studies of the lesional skin from mice revealed significant hyperplasia of the epidermis in the Con-AsE group compared with the HI-AsE-and vehicle-treated groups (Figure 8C). Epidermal thickness of the Con-AsE group was also significantly greater than that of the HI-AsE-and vehicle-treated groups ( Figure 8D). These findings demonstrate that HI-AsE does not sensitize iScience Article or trigger pathogenic reactions in the skin or lungs, thus enhancing the applicability of HINS materials in daily life for both breathing apparatus and skin-contact-related home amenities. Collectively, our data confirmed that the use of HINS composites within the safe concentration range could not only inhibit the growth of hyphae and reduce the pathogenic source but also decrease the use of antibiotics for prophylaxis or treatment of invasive aspergillosis.

Conclusion
In our study, we stimulated the skin and lung tissues with the AsE antigen for the first time, simulating the Aspergillus-induced skin and lung hypersensitivity animal model. Currently, many research groups are working on optimizing the performance of ZnO by the surface modification methods. Among these, studies on biomolecular modification with the benefits of biocompatibility and nontoxicity are promising in therapy applications. This work has provided a strong framework for future studies on the mechanism of Aspergillus sensitization. First, pneumonia and pneumoniaÀskin disease complication models were induced successfully with the safe and non-active AsE. Compared with the traditional dust induction method, it is easier to control the state of lesions to achieve guiding significance. Second, our in vitro experiments observed that the HINS complex successfully inhibits the fungi growing at a 5.0% mixture ratio for the HINS-treated agar, and with the elongation of the treatment time, the effect of arresting the mycelium was significantly noticeable. Drug resistance development and pathogenicity are associated with biofilm formation by fungal pathogens. In this context, we believe that as a prophylactic measure against pathogenic fungi, promoting the HINS composite substance for packaging of daily stuff, construction materials, and medical instruments (Scheme 1) could provide clean auxiliary medical equipment for patients undergoing disease treatment. Furthermore, we discovered that HI-AsE does not trigger any significant allergic response in mouse lung and skin tissues by analyzing and comparing iScience Article extracts collected from Con-AsE-treated and HI-AsE-treated mice. This may be because of the inhibition of the development of toxicity in the hyphae of Aspergillus by HINS composites. Through gene expression analysis (tpsA, tpsB, tpsD, gel4, aspA, hsp90, and dprA) in the Hi-AsE and Con-AsE groups, we showed the HINS composite inhibited growth of hyphae, which would attenuate the hypersensitivity responses. Moreover, the HINS composites show high biological compatibility with few side effects when used in a safe concentration and thus could be used in medical materials and devices to provide clean auxiliary medical equipment for patients undergoing disease treatment.
The mycelium tip shows a gradient growth pattern, and the mycelium tip expands the fastest. Hyphal swelling and numerous hyphae were key features of Aspergillus cultured in common nutrition agar and may be indicative of mycotoxin biosynthesis and pathogenicity. Using the HINS composite within the safe concentration range could not only inhibit the growth of hyphae but also reduce the number of fungal pathogens. However, the industry-scale production and stability of HINS are understudied, and the durability of its effectiveness is an interesting topic for future study. We believed that the HINS would be used in producing safety equipment, kits, with prophylactic and therapeutic agents against pathogenic fungi. We envision the HINS composites will be used in antibiotic applications combined with the research on the physical and chemical properties of theriacs providing the challenges of studying the functional mechanism of HINS and other nanomaterials for further applications, including the protection of genetic diversity and medical safety, are addressed.

Limitations of the study
Like every coin has two sides and the effect of microbes on humans will always be an ambiguous topic without a precise definition. On the one hand, human immune responses to fungi are intricate, starting from innate (non-specific) immune reactions to particular acquired immune responses induced during pathogen infestations, which have encouraged the study of human antifungal immune reactions and the respect for biodiversity. On the other hand, controlling fungi infestations requires understanding the growth and pathogenic mechanisms. Our model remains unable to fully reflect actual fungal skin exposure in a normal human environment in terms of viability, amount, and life cycle of Aspergillus; optimization of this experimental approach in a future study would facilitate further research on pathogenic mechanisms and provide constructive experimental evidence for the development of therapeutic drugs. Meanwhile, the study on genetic and histological analysis of infectious mouse model is quite novel which encourages us to pay more attention to studying their specificity and immune mechanisms. 10. Ghosh, A., Sarkar, A., Paul, P., and Patel, P.

Materials availability
Materials are available up on request.

Chemicals and reagents
All experimental reagents were analytically pure and used without further purification.

Biological samples
A. fumigatus ATCC 36607 was grown in Sabouraud dextrose agar at 25 C for 5 days. After culture, the suspension was centrifuged at 1,400 g for 10 minutes. The pellet of the Aspergillus fungus was re-suspended in PBS and quantified using a hemocytometer. The HINS treatment Aspergillus were collected using three different methods (À56 C dry ice and dried at room temperature [RT] for live Aspergillus, autoclaved at 121 C for 15 min for non-active Aspergillus). To test the effect of HINS nanomaterials on fungi, we prepared the glucose agar medium plates and the specific solid medium. Fungal spores after 5-day culture were collected (600 spore/20 mL) in PBS solution to obtain uniformity and reproducibility in the experiment. The 20 mL of spore seed solution has been dropped uniformly on the center of the agar medium plates with the different concentrations of HINS, dried, and placed upside down. The plate was kept at 36 C for 3 days, and a photographic record of each fungal sample was taken. Image-J was used to analyze the records to compare the growth rate of each HINS samples with the control sample.

Preparation of the HINS composite
To enhance the stability and biocompatibility of ZnO-NS, a homobifunctional imidoester (HI, DMS) modification was carried out according to the previous study. 26 Briefly, 4 mg ZnO-NS (40 mg/mL, 100 mL) and 4 mg HI (DMS, 10 mg/mL, 400 mL) were dissolved in a 1.5 mL EP tube in a 500 mL solution. The mixture was oscillated by an oscillating machine (Magic-mixer TMM-5). After 20 min, the mixture was centrifuged at 12,000 rpm for 5 min in a mini-centrifuge (LaboGene). The supernatant liquid was removed, and the precipitate was washed twice with Milli-Q water. Finally, 400 mL of Milli-Q water was used to re-suspend the precipitate to obtain 10 mg/mL of HINS composite solution. The precipitate was dried overnight in a drying oven (DX312C, Yamato) at 56 C. iScience Article of 4% SDS. After 2 h, the shaved dorsal skin surface was treated with 40 mg of dried non-active Con-AsE or HI-AsE. The negative control group was coated with PBS only as a vehicle. Inductions were performed 3 times per week for three consecutive weeks.

Evaluation of dermatitis severity
The scoring atopic dermatitis (SCORAD) index was used in mice to assess the severity of dermatitis symptoms. 46 This index is determined by the presence of erythema/bleeding, scarring/dryness, edema, and flaking/erosion. Each symptom is assigned a score of 0 (none), 1 (mild), 2 (moderate), or 3 (severe). The total SCORAD score is the sum of each individual's scores. 47 Two independent researchers evaluated each score on a weekly basis. Pictures were captured with the same camera settings and lighting every week.

Histological analysis for lung tissue and skin tissue
For histological examination, lung tissue was removed and fixed in a 10% (v/v) neutral-buffered formalin solution. Lung tissue was embedded in paraffin. Sections were cut at 4 mm thickness and stained with H&E and PAS. 5,48 The extent of inflammatory cell influx, mucus production, and goblet cell invasion was observed under a light microscope (Magnification, 3100 and 3200). Peribronchial infiltration and goblet cell hyperplasia were assessed by semi-quantitative scores (0-5), as reported previously. 49 To grade peribronchial inflammatory cell infiltration, the peribronchial cell count was determined based on the following 5-point grading system: 0, normal; 1, few cells; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells of 2-4 cells deep; 4, a ring of inflammatory cells of more than 4 cells deep. 50,51 The extent of mucus production was assessed by PAS staining, and goblet cell hyperplasia in the airway was scored as follows: 0, no goblet cells; 1, <25%; 2, 25-50%; 3, 50-75% (including 50%); 4, >75%. Five-to-six fields of view were counted per stained tissue sample, and the mean score was calculated for 5 mice per group. Measurements were scored by 2 experimenters in a blinded manner. The dorsal skin tissue of each group of mice was cut with a sterile scalpel and fixed with 4% paraformaldehyde for H&E staining. Epidermal thickness was measured by Meta-Morph image analysis software (Molecular Devices, Sunnyvale, CA, USA), and eosinophils were counted using an Olympus BX53 microscope (Olympus Corp., Tokyo, Japan). Three different images were taken from each slide. 46 Measurement of sera immunoglobulins by enzyme-linked immunosorbent assay (ELISA) Whole blood was collected from mice before sacrifice, and serum samples were collected by centrifugation at 4,000 rpm, 4 C for 15 min. Total immunoglobulin E (IgE) levels in mouse serum were measured from an initial serum dilution of 1/10 in PBS+1% bovine serum albumin using an ELISA kit (BioLegend, San Diego, CA, USA, Cat #432404) according to the manufacturer's instructions.